In the process of analytical analysis of fluid samples (biologic samples, chemicals reagents, and gases) it is common for test samples to be passed through a chamber containing either a detection substrate, or a transparent window allowing the interrogation of the sample by some form of energy or light. It is common for sample fluids to be delivered and removed from these “detection chambers” using a continuous flow of transport fluid entering the chamber from one end and exiting the chamber at another. Thus these chambers are termed detection “flow cells”, and the analysis techniques that utilize them are termed “flow based” detection methods. During flow based analysis, sample fluids to be tested are delivered as discrete volumes, or ‘plugs’, within a stream of continuously flowing buffer passing through the flow cell and over the detection substrate. The accuracy, sensitivity, and applicability of flow based analysis techniques are highly dependent upon the process and characteristics of the sample fluid delivery to, and removal from, the detection flow cell.
Researchers in a wide variety of fields such as medicinal science and environmental analysis, to name just a few, need to characterize the interactions of biologic molecules found in human, animal, or plant fluids and tissues. These characterizations commonly involve bringing two or more different types of sample molecules into physical contact with each other for a set period of time and then measure if, for example, they have combined to form a molecular complex, or if either has caused a change to the physical structure or function of any of the other reactants. Understanding the kinetics (speed) and affinity (strength) of these molecular interactions are just two of the parameters often measured during these characterization procedures, termed ‘molecular interaction analyses’. Typically when utilizing flow cell based analysis techniques during molecular interaction analysis, a population of one of the interacting molecules is permanently attached, or ‘immobilized’, onto the detection substrate or window within flow cell. Sample containing the other molecule(s) to be investigated are then passed through the flow cell so they have the opportunity to interact with the immobilized molecules and those interactions measured.
So called biosensors, or “label-free” analysis techniques, commonly utilize detection flow cells and flow based sample delivery methods to “present” test samples to be analyzed to the detection sensor surface or substrate. The use of flow based sample delivery in label-free biosensor instruments can greatly increase the amount of information these techniques can generate about the molecular interactions being investigated. Biacore instruments sold by GE Healthcare are a well known example of label-free analytical biosensors used in biological research for molecular interaction analysis studies. In the case of Biacore instruments, an optical detection technique called Surface Plasmon Resonance (SPR) is employed to measure mass changes on metal surfaces. These mass changes on the sensor surface result from the addition or subtraction of molecules onto the surfaces due to the interaction of molecules with either the sensor surface itself or another molecule attached to the surface. Other examples of analysis techniques that characterize molecular interactions using label-free detection methods include Dipolar Interferometry, Quartz Crystal Microbalance (QCM), Surface Acoustic Wave (SAW), and micro-cantilevers. Aside from eliminating the additional analysis steps, reagents, and sample preparatory requirements of label based testing methods (RIA, ELISA, and Fluorescence techniques), label-free analysis enable the measurement of the molecular interactions under investigation to be recorded as they occur. These real-time analysis capabilities have the potential to provide a great deal of information in addition to confirming the specific binding of target molecules, as is arguably the only capability of label based techniques. Under the proper conditions, real-time, label-free analysis techniques have the ability to determine the speed and strength of molecular interactions, and in some cases, if those interactions resulted in any structural changes to the test molecules. But it has been well documented that these real time analysis capabilities, as well as the accuracy, and sensitivity of label-free detection techniques in general, are highly dependant on the quality of the corresponding flow based sample delivery methods.
For example, one critical aspect of sample delivery in flow cell based analysis techniques is the fast and efficient transition from one reagent to the next within the flow cell. This need for fast and efficient transition between reagents is most clearly demonstrated when characterizing molecules that exhibit very low binding affinity (weak in ‘strength’) for one another. The association rates (molecules coming together), and dissociation rates (falling apart), termed “kinetic rates”, associated with these low affinity interactions often occur within the first few seconds after the test molecules are brought into contact with one another or separated. Thus, the capability to obtain accurate measurements just after the test molecules have come into contact, and immediately following their separation, is crucial to accurate kinetic rate characterization of low affinity molecular interactions.
During automated testing procedures using flow cells, it is commonly advantageous for liquid handling devices to transfer the sample volumes to be analyzed from their storage containers or vials to the chamber or detection flow cell as a plug volume pushed through tubing pathways by another liquid termed the running buffer. As the plug volume of sample liquid is pushed through the tubing of the liquid handling unit, mixing between the plug and the running buffer will often occur creating a volume of liquid at the front and back of the sample plug that is a variable gradient of sample and running buffer. As the concentration of this mixture is unknown, including it in the final analysis of the sample can often interfere with the accuracy and sensitivity of testing.
Thus, it is common for a “cutting” event to be performed on the sample plug volume just prior to its introduction into the analysis chamber. These cutting events typically involve some initial portion of the sample plug volume being directed to a waste just prior to the sample analysis process. Often mechanical valves are used to perform this function but due to limitations in valve technology related to sample waste, valve dimensions, and poor robustness, these structures and methods are not ideal.
Additionally, as the reagent plug enters the flow cell it pushes assay buffer out, with the reverse occurring at the end of the plug injection. During this process, a period of transition occurs where the flow cell, and thus the detection substrate, is exposed to a concentration gradient or mixture of sample and buffer. During these ‘transition periods’, accurate determination of kinetic rates is not possible as the true concentration of test sample exposed to the detection surface is unknown. Thus, the ability to quickly switch from one fluid to the next within the flow cell during analysis, i.e., the delivery of highly discrete volumes of sample fluid having a clean leading edge without a concentration gradient within a continuous flow of transport fluid, is critical to obtaining as much usable data as possible.
The vast majority of current flow based sample delivery technologies, even on a micro-fluidic level, do an inadequate job of efficiently transitioning between samples or sample and buffer. It is not uncommon for microliters and even ten's of microliters of fluid to pass over the detection surface before contacting solution that is 100% test reagent. As typical test volumes can be less than fifty microliters, flowing at ten's of microliters per minutes, these long transition times severely affect measurement capabilities. The long transition times are mainly due to the physical design of valve technology built into the sample delivery systems, which can often only be effectively utilized at some distance from the flow cell and detection surface. Thus the reagent plug must travel a distance before contacting the detection surface, during which reagent solution mixing will occur. Microfluidic tubing designs employing micro valves have been used with moderate success to overcome this situation as they minimize liquid travel and the micro valves can be located much closer to the detection flow cell. But, due to their design and small size, these valves are costly, often mechanically unreliable, and susceptible to clogging.
Another critical aspect of sample delivery in regards to kinetic rate analysis is the ability for sample molecules to efficiently diffuse from the sample plug onto the sensor surface as the sample plug passes over. It has been well documented that inefficient transport of sample molecules to the sensor surface, termed “mass transport limitations”, results in inaccurate estimations of kinetics rates. Efficient molecular diffusion from the sample plug to detection surface is facilitated by passing the sample over the detection substrate as quickly as possible (i.e. fast sample flow rates). But when considering the practical applicability of flow cell based analysis techniques, the requirement to pass sample over the detection surface at high rates of speed becomes a liability.
As the physical nature of molecular interactions often means that sample molecules must be in contact for several minutes to obtain accurate measurements, high sample flow rates during analysis result in the consumption of large volumes of test sample. Historically the most common way to lower sample volume requirements while maintaining high analysis flow rates has been to minimize the size of the detection flow cells. But due to a variety of issues related to the different detection technologies (i.e. size of the detection substrates, electronics, and optics), and the need to interface those technologies with high performance and robust sample fluid delivery systems, there have been practical limitations to the miniaturization of detection flow cells. Thus, with the resource requirements to produce even the crudest biologic samples for testing being very high, and the fact that the new research disciplines such as Proteomics continue to expand the number of samples to be evaluated, there is an ever increasing demand to work with the smallest sample volumes possible.
The next critical aspect when evaluating the applicability of a technology for molecular interaction analysis is the requirement to simultaneously evaluate large numbers of samples while still meeting the requirements of delivering highly discrete, and small volumes of sample at high rates of flow. This process of simultaneous multi-sample analysis is often referred to as High Throughput Sampling, or HTS. Often, based on the analysis methods used in conjunction with HTS, there is a desire in some instances to handle each sample analysis as a completely independent procedure, and in other instances to handle the multiple analyses using exactly the same procedure and reagents. Thus the ultimate applicability for high throughput analysis comes when the user can switch between “individual” and “common” processing of the multiple sample analyses at any time during the testing procedure. Often these variations in testing procedures represent nothing more than different reagents being applied to different test vessels at certain stages of the testing process. For test methods that employ the analysis of molecules coated onto an array surface, this process of individual and common handling of the multiple individual analyses becomes a process of individual and common “addressing” of different reagent fluids to the different locations of the array. In some steps of the assay procedure it is preferable that the same reagent can be addressed to more than one or all of the target locations on the array. In other cases it is desirable to address a different reagent onto each target location.
In the past, a variety of techniques based on the manipulation of the process of Hydrodynamic Focusing have been employed in an attempt to address these requirements. The so called, “Hydrodynamic Addressing” and “Hydrodynamic Guiding” techniques, use guide fluid streams to position sample fluid streams over different sections of array surfaces within flow cell chambers.
One example of a technique of this type is shown in published PCT Publication No. WO/2003/002985, which is incorporated by reference herein and as shown in FIGS. 1 and 2, discloses a method of operating an analytical flow cell device comprising an elongate flow cell having a first end and a second end, at least two ports at the first end and at least one port at the second end, comprises introducing a laminar flow of a first fluid at the first end of the flow cell, and a laminar counter flow of a second fluid at the second end. Each fluid flow is discharged at the first end or the second end, and the position of the interface between the first and second fluids in the longitudinal direction of the flow cell is adjusted by controlling the relative flow rates of the first and second fluids. Also disclosed are a method of analyzing a fluid sample for an analyte, a method of sensitising a sensing surface, and a method of contacting a sensing surface with a test fluid.
Another example is found in PCT Publication No. WO/2000/056444 that is also incorporated by reference herein and as shown in FIG. 3, illustrates a composition of a liquid (26) that is caused to interact with a narrow band shaped area at a chosen position on a solid surface within a flow channel (12) by hydrodynamic focusing of a guided stream of said liquid between two streams of guiding liquid (28). By altering the ratio of the flow rates of the two guiding liquid streams, the position of the guided liquid stream is changed and further interaction with the solid surface takes place along a second band shaped area. Using two such flow channels it is possible to produce a two dimensional array of interaction sites.
Still another example is disclosed in PCT Publication No. WO/2006/050617 which is incorporated by reference herein and illustrates in FIGS. 4a-4g a microfluidic device and its use for the production of micro-arrays, in particular for the detection of protein interactions, is described. The microfluidic device comprises a flow cell part (1) and a chip part (2) together forming at least two crossing, preferably perpendicular, closed channels (3, 4), said flow cell part forming open channels providing the bottom wall and at least part of the side walls, in particular three walls of said closed channels (3, 4), said closed channels (3, 4) being connected to at least three fluid providing means for generating at least three fluid flows (7) and said closed channels (3, 4) being designed and dimensioned such that the flow of at least three aqueous fluids streaming through each of said channels (3, 4) is laminar at least until after said crossing of said channels (6), said chip part (2) forming the top wall and optionally part of said side walls, in particular the fourth wall, of said closed channels (3, 4) and having a surface that is activatable by reaction with an activating molecule.
However, these prior art techniques and structures shown in FIGS. 1-4g are limited to addressing sample fluid streams in single dimensions within the array. Thus, if a surface array is viewed as an x-y grid, these techniques can either address only the entire x-row or the entire y-column with a single reagent. These techniques offer no remedy to address individual x-y locations, or “spots”, on the array independently severely limiting the flexibility of array design. Thus it is desirable when working with array based testing methods to have the ability to address each test location on the array as a completely individual entity in some instances, and in other instances to treat more than one or all of the test locations in the same manner.
In summary, there remains a considerable need for greater control and flexibility in regards to the volume, speed, and location of reagent presentation to detection surfaces in flow cell based analytical testing technologies.